Cardiovascular disease is the leading cause of death in the United States. In women, the incidence of cardiovascular disease increases after menopause. A primary risk factor for cardiovascular disease is hypertension, which also increases in prevalence following menopause. Nitric oxide (NO) induced vasodilatation is reduced with age, resulting in increased blood pressure and reduced limb blood flow. However, postmenopausal women on hormone replacement therapy (HRT) have improved NO induced limb blood flow response at rest. The effect of estrogen on the content of endothelial nitric oxide synthase (eNOS), the key enzyme in NO production, has not been investigated in vivo in humans. This is despite in vitro evidence that eNOS content is increased in response to estrogen exposure. We hypothesize that there are reductions in eNOS expression and nitric oxide dependent skeletal muscle blood flow following menopause, and that these reductions can be counteracted, or reversed, by estrogen hormone replacement therapy. We will investigate 25 premenopausal (20-30 yr) sedentary women, as well as 50 postmenopausal (60-70 yr) sedentary women before and after eight weeks of HRT or placebo. A muscle biopsy will be taken to measure eNOS content, and nutritive skeletal muscle blood flow will be monitored using microdialysis at rest and during cycle ergometry exercise. These investigations will determine if HRT improves nitric oxide dependent nutritive blood flow in skeletal muscle at rest and during exercise, and will determine if this improvement is associated with increases in eNOS in skeletal muscle biopsy samples. The long-term objectives of these investigations are: 1) to identify mechanisms responsible for a reduced nitric oxide-dependent vasodilatation in postmenopausal women and 2) to find practical means of reversing vasodilator decrements in postmenopausal women, thereby reducing the incidence of cardiovascular-related diseases in this population. These investigations will provide valuable research exposure to our undergraduate and graduate programs, and will provide data for subsequent R01 applications.